In the course of investigating the reported acid phosphatase activity of carbonic anhydrase III, several contaminating phosphatases were discerned. One of these, a magnesium-dependent species of 18.6 kilodaltons, has been purified to homogeneity and has yielded several peptide sequences from which the parent gene has been identified by database searching. Although orthologous genes can be identified in fungi and plants, as well as in mammalian species, there is no apparent homology to any known family of phosphatases. In fact, no homology to any protein of known function can be convincingly demonstrated. In recognition of this fact, the enzyme has been given the name Magnesium- Dependent Phosphatase-1 (MDP-1). One representative mouse cDNA was expressed in E. coli and purified. Similarly, the homologous S. cerevisiae gene was cloned from yeast genomic DNA, expressed, and purified. Both enzymes show magnesium-dependent acid phosphatase activity comparable to the originally isolated rabbit protein, confirming that the sequenced protein is, in fact, the active species. In the absence of sequence homology, the question of classification of this phosphatase is less than straightforward and hinges on two primary concerns: what is the nature of the catalytic mechanism and what is the nature of the substrate? We have thus far shown that the enzymatic mechanism is independent of cysteine or histidine residues and, aside from its requirement for magnesium, does not utilize any other metals. Thus, MDP-1 is either (a) a metalloenzyme utilizing only magnesium where an activated water molecule is the nucleophile, (b) a member of the class of phosphatases utilizing aspartic acid as a nucleophile (many of which are magnesium-dependent), or (c) representative of a new class of phosphatase. Concerning possible substrates, we have shown that MDP-1 is specific for phosphotyrosine over phosphoserine and phosphothreonine and is capable of hydrolyzing the phosphate from phosphotyrosine-containing polypeptides. Thus, MDP-1 may represent a new class of protein-tyrosine phosphatase (all others utilize a cysteine-dependent mechanism). Although MDP-1 also shows a preference for ribose-5-phosphate over glucose-6-phosphate, nucleotide monophosphates are poor substrates and thus a role in RNA or DNA biochemistry seems unlikely. - phosphatase, new, enzyme, tyrosine, magnesium